The Enhancer of zeste [E(z)] locus of Drosophila melanogaster is implicated in multiple examples of gene repression during development. First identified as dominant gain-of-function modifiers of the zeste1-white (z-w) interaction, mutant E(z) alleles also produce homeotic transformations. Reduction of E(z)+ activity leads to both suppression of the z-w interaction and ectopic expression of segment identity genes of the Antennapedia and bithorax gene complexes. This latter effect defines E(z) as a member of the Polycomb-group of genes. Both maternally and zygotically produced E(z)+ activity is required to correctly regulate the segment identity genes during embryonic and imaginal development. Complete lack of zygotically produced E(z) activity results in blockage of cell proliferation in larval neuroblasts, and disruption of chromosome organization. Thus, it seems that reduction of E(z)+ interferes with multiple examples of gene repression, and that its complete absence blocks cell proliferation and disrupts chromosome organization. Therefore, through molecular and biochemical analysis of E(z) protein activity, a link between gene regulation and higher order chromatin organization may be elucidated. The long term goal of this work is to gain an understanding of the molecular mechanisms by which the E(z) product, and those of other Polycomb-group genes, carry out their genetically defined functions. The specific aims of this proposal are to utilize a multifaceted approach including genetic, molecular, immunological and biochemical tools to (1) determine the localization of the E(z) protein, both subcellular and within the whole organism, (2) elucidate the molecular mechanism(s) by which it represses transcription, and (3) dissect the functional domains of the E(z) protein. The approaches to be taken include (1) use of antibodies specific for the E(z) protein to determine its localization, (2) use of these same antibodies to examine potential in vitro and in vivo interactions of the E(z) protein both with regulatory DNA sequences that have been identified as targets for E(z) activity, and with the products of other genes that have been genetically shown to interact with E(z), and (3) DNA sequence analysis of existing mutant E(z) alleles, and in vitro construction of altered forms of the gene, which will then be both expressed in vitro and introduced into the Drosophila germ line and assayed for in vitro and in vivo activities, respectively. Misregulation of gene expression has been implicated in a number of diseases, including various cancers. In the long term, a better understanding of the mechanisms by which genes are regulated and the potential role of higher order chromosome organization will be crucial to understanding and treating such diseases.